Application of the automated haematology analyzer XN-30 in an experimental rodent model of malaria

C57BL/6 and ICR mice (6 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). For parasite infection, the mice were injected intraperitoneally with 3 × 10⁵ iRBCs with the non-lethal strain, P. yoelii 17XNL, suspended in phosphate-buffered saline (PBS).

Standard thin blood smears were fixed in 100% methanol (Nacalai Tesque, Kyoto, Japan) for 10 min and then stained in 10% Giemsa stain working solution, pH 7.2 (Merck KGaA, Darmstadt, Germany) for 13 min. The slides were observed at 1000× magnification using a BX50 light microscope (Olympus, Tokyo, Japan). Parasitaemia was determined by calculating the ratio of iRBCs in at least 3000 RBCs.

FCS files exported from the XN-30 analyzer were analysed by Flowing software 2.5.1 (Turku Centre for Biotechnology, University of Turku, Turku, Finland). Areas of iRBCs, polychromatic RBCs, HJB-RBCs, and WBCs were gated on the M scattergrams and areas of iRBCs, HJB-RBCs, and merozoites were gated on the M(SSC-FSC) scattergrams. Common dots in iRBC areas on both the M and M(SSC-FSC) scattergrams were assigned as iRBCs and counted (see Fig. 2b; red dots). This method using SFL and FSC parameters for M scattergrams, and SSC and FSC parameters for M(SSC-FSC) scattergrams was designated as ‘three-dimensional analysis’. Each scattergram represented dots per 0.953 μL and the analyzer reported the number of total RBCs per μL. This difference was compensated in the calculation of parasitaemia, as shown in following equation:

RBC, WBC, and PLT counts, as well as HCT value were calculated according to the indicated dilution ratio; MCV and MPV values were directly adopted.

The re-analysis of the scattergrams from the XN-30 analyzer by using Flowing software is referred to as the ‘XN-30 system’ in this study.

As stock solution, artemisinin (TCI, Tokyo, Japan) was dissolved at 50 mg/mL in 65% dimethyl sulphoxide and 35% Tween-80. The stock solution was further diluted to 5 mg/mL (1:10 dilution) with saline (Otsuka Normal Saline; Otsuka Pharmaceutical Co Ltd, Tokyo, Japan). Mice were subcutaneously injected with artemisinin (25 mg/kg) 4 days after infection with the P. yoelii 17XNL parasite. Blood samples were collected before and after drug treatment and analysed using the XN-30 system (see also Fig. 5a).

The correlation between parasitaemia determined using the XN-30 system and by microscopy was analysed using regression analysis. The coefficient of determination (R²) was calculated using Microsoft Excel (Microsoft, Redmond, WA, USA). Mean, standard deviation (SD), and coefficient of variation (CV % = SD/mean × 100) were calculated using Microsoft Excel (Microsoft) and Graphpad Prism version 5.0 (Graphpad Software, San Diego, CA, USA). The statistical significance of differences between non-infected and infected groups was evaluated through one-way analysis of variance followed by Dunnett’s multiple comparison tests using Graphpad Prism version 5.0 (Graphpad Software).

As a first step to apply the XN-30 analyzer for the detection and measurement of iRBCs in mouse, blood samples from non-infected healthy mice were measured. Signals from non-diluted blood samples were saturated (100%, Additional file 1: Figure S1a, b); however, the blood samples diluted 10 to 50-fold were adequately represented on the M scattergram (female, Additional file 1: Figure S1a; male, Additional file 1: Figure S1b). For healthy human peripheral blood samples, the XN-30 analyzer distinguished WBCs (1) and polychromatic RBCs (2) on the M scattergram (Fig. 1a(i)); however, HJB-RBCs (3) were also detected for the mouse peripheral blood samples. Unfortunately, HJB-RBCs were recognized as ring-forms, trophozoites, and polychromatic RBCs (Fig. 1a(ii) and Additional file 1: Figure S1a). These findings suggest that the optimal working dilution for the samples needs to be determined and that HJB-RBCs hindered the precise enumeration of iRBCs. Moreover, as the XN-30 analyzer simultaneously reports other parameters such as RBC, WBC, and PLT counts, as well as HCT, MCV, and MPV values [7], the reliability of the data from diluted blood samples was also evaluated by comparing the counts and values of these parameters across varying dilutions. The comparison indicated that the dilutions hardly affected the haematology results (female, Fig. 1b; male, Additional file 1: Figure S1c). From these data, it can be inferred that 2 μL (1:50 dilution) of blood sample is sufficient for suitable measurements.

The XN-30 analyzer showed WBCs, polychromatic RBCs, and HJB-RBCs in non-infected mouse blood samples on the M scattergram (Fig. 2a, upper left panel), similar to that in Fig. 1a(ii). The M(SSC-FSC) scattergram using the SSC parameter instead of the SFL parameter revealed the separation of these cells (Fig. 2a, upper right panel). However, HJB-RBCs were still misrecognized as ring-form parasites (Fig. 2a, upper panels). In infected blood samples, the analyzer detected iRBCs and HJB-RBCs, but was not able to distinguish the different developmental stages of the parasite (especially the trophozoite and schizont stages), owing to high multiple infection (Fig. 2a, lower panels and Additional file 1: Figure S4). Moreover, polychromatic RBCs and HJB-RBCs were again misrecognized as trophozoite and ring-forms. Thus, the data obtained from the analyzer require further analysis to resolve these inherent limitations. To overcome these issues, the scattergrams were analysed using the Flowing software in the XN-30 system described in the “Methods”.

Manual gating distinguished polychromatic RBCs, HJB-RBCs, and WBCs from iRBCs on the M scattergram; however, any dots in the area of the iRBCs were counted as iRBCs on the M scattergram, even in non-infected blood samples (Fig. 2b, upper panels). For infected blood samples, merozoites can be resolved in addition to HJB-RBCs on the M(SSC-FSC) scattergram (Fig. 2b, lower panels). Common dots in the iRBC areas on both the M and M(SSC-FSC) scattergrams were assigned as iRBCs. This analysis showed that the iRBC counts were 11 and 9686 (parasitaemias were 0.0057% and 6.7%, respectively) in the non-infected and infected blood samples, respectively (Fig. 2c); with 0.0057% of the parasitaemia in the non-infected blood sample regarded as false-positive. Further analysis of non-infected blood samples from female and male mice revealed false-positive parasitaemia values of 0.0043 ± 0.0013% and 0.011 ± 0.0057%, respectively (Additional file 2: Table S1). Of note, for infected blood samples, the 6.7% parasitaemia from the XN-30 system was similar to the 5.5 ± 0.35% parasitaemia determined through microscopy (Fig. 2c, lower line). These results indicate that the three-dimensional analysis using the SSC parameter added to the FSC and SFL parameters (see “Data analysis by Flowing software” in “Methods”) permitted a more accurate determination of iRBC count in mouse blood samples.

To further test the reliability of the XN-30 system, 5 mouse blood samples with different parasitaemias were compared using the XN-30 system and microscopy. The XN-30 system had less dispersion in a single sample (CV% = 2.36–2.96 in the XN-30 system; 3.14–18.93 in microscopy) and larger mean parasitaemia (XN-30/microscopy = 1.02–1.25) than that observed through microscopy (Fig. 3a, Additional file 1: Figure S2 and Additional file 2: Table S2). However, correlation analysis revealed a high coefficient of determination (R² = 0.998) between the two (Fig. 3b). With these results, XN-30 system is potentially more reproducible and sensitive than microscopy. Furthermore, in addition to the parasitaemia, it was observed that RBC, WBC, and PLT counts, as well as HCT, MCV, and MPV values were reliable even using diluted samples (see Fig. 1b). When infected, with parasitaemia, haematological parameters fluctuated as expected (Fig. 3c, also see below).

The course of infection was monitored using both the XN-30 system and microscopy. Both methods showed increase in parasitaemia until day 14–17 after which parasitaemia declined (Fig. 4a and Additional file 2: Table S3). A higher mean parasitaemia was obtained with the XN-30 system than with microscopy (Fig. 4a and Additional file 2: Table S3), similar to the results presented in Fig. 3a and Additional file 2: Table S2. A correlation analysis indicated a strong coefficient of determination (R² = 0.973) (Additional file 1: Figure S3), as mentioned above (see Fig. 3b). Likewise, in addition to parasitaemia, the XN-30 analyzer was able to evaluate other parameters. RBC count and HCT value decreased as parasitaemia increased, and both values returned to baseline as parasitaemia is cleared (Fig. 4b(i), (iv)). This fluctuation matches with the anaemia status of mice after infection. However, WBC fluctuation differed largely in individual mice (Fig. 4b(ii)), rising rapidly after infection and decreasing around 14 days when parasitaemia is at its peak. The PLT count decreased as parasitaemia increased; however, its return to baseline was delayed compared with that of the RBC count and HCT value (Fig. 4b(iii)). Fluctuations in MCV and MPV values followed those of parasitaemia (Fig. 4b(v), (vi)). An increase in MCV and MPV values corresponded with the enlarged morphologies observed by microscopy (Additional file 1: Figure S4). The comparison of the iRBC count (iRBCs/μL) with parasitaemia (%) revealed that iRBC levels plateaued at approximately 3.0 × 10⁶ iRBCs/μL when the parasitaemia was approximately more than 20% (Fig. 4c). This finding indicates that the increase in parasitaemia for P. yoelii 17XNL was dependent on reductions in the levels of total RBCs greater than 20% parasitaemia. To validate this finding, RBC counts were correlated with parasitaemia. The comparison revealed a logarithmic negative correlation (Fig. 4d), suggesting that the total RBC levels were logarithmically reduced after infection and that the maximum concentration of iRBCs is regulated by uncharacterized mechanisms in mice and/or parasites.

To evaluate the efficacy of anti-malarial drugs with the XN-30 system, a rodent model for malaria was used to analyse parasite-infected mouse blood before and after artemisinin treatment. Mice were infected with the parasite on day-4 and then treated with artemisinin or solvent (Fig. 5a). Parasitaemia promptly decreased after artemisinin treatment (Fig. 5b; red lines) while it increased in solvent-treated mice (Fig. 5b; blue lines). Haematological parameters fluctuated in both artemisinin- and non-treatment groups. With artemisinin, treatment resulted in the recovery of RBC, PLT counts and HCT value, while WBC count, MCV, and MPV values barely fluctuated from baseline (Fig. 5c). From these results, XN-30 analyzer permits, aside from parasitaemia determination, the haematological evaluation in vivo and provide clues as to the curative effects of anti-malarial drugs.